Myelination gets direct

Separate groups at the Case Western Reserve
University School of Medicine and the Stanford University School of Medicine have
developed similar approaches to directly reprogram rodent fibroblasts into
oligodendrocyte progenitor cells.1,2 The direct lineage conversion
method could be a safe and fast way to supply cells for myelination disorder
cell therapies. Now, the teams need to show that their methods also reprogram
human fibroblasts.

Myelination
disorders include leukodystrophies and autoimmune conditions such as multiple
sclerosis (MS). In all cases, loss of the myelin sheaths around axons of
neurons impairs nerve firing and causes nervous system deficiencies.

Most
available treatments for myelination disorders aim to slow demyelination or
treat symptoms, but none repair or replace myelin.

One
starting point for that goal is oligodendrocyte progenitor cells (OPCs), which
are components of the CNS white matter that generate oligodendrocytes, the
cells that produce myelin during development and after injury in the CNS.

But
current sources of OPCs are limited to donor and embryonic stem cell tissues,
both of which carry risks of immune rejection by the recipient. Donor OPCs also
are in short supply, and cells derived from human embryonic stem cells come
with a host of ethical issues and concerns about potential teratogenicity.

As
an alternative, several groups including a team at Case Western have derived
OPCs from rodent fibroblast-derived induced pluripotent stem (iPS) cells.3
iPS cells would be patient specific but require multiple manipulation steps,
and the strategy has not yet yielded human OPCs.

The
most recent approach for cell differentiation is direct lineage conversion-reprogramming
somatic cells into a desired cell type-which eliminates many manipulation
steps.

The
Stanford team is among the groups that have successfully reprogrammed rodent
fibroblasts into neurons and neural stem cells.4

Now,
the Case Western and Stanford teams have accomplished direct lineage conversion
of mouse fibroblasts into OPCs. In papers published in the same issue of Nature
Biotechnology, the teams used forced expression of OPC-specific
transcription factors to reprogram rodent fibroblasts into OPCs that generated
functional, myelin-producing oligodendrocytes.

The
Case Western team was led by Paul Tesar, assistant professor of genetics and
genome sciences. His group transfected mouse embryonic fibroblasts with a pool
of lentiviral vectors carrying eight doxycycline-inducible
transcription factors that were highly expressed in OPCs and known to play
roles in oligodendrocyte development.

In
culture, fibroblasts were reprogrammed with the transcription factor pools into
cells that expressed OPC lineage genes and suppressed fibroblast-related genes.
The cells differentiated into myelinating oligodendrocytes.

In
cultured brain slices from early postnatal mice with hypomyelination due to
loss of myelin basic protein (Mbp), transplantation of the reprogrammed
OPCs with doxycycline led to myelination of the axons.

Transplantation
of the reprogrammed cells into the dorsal spinal column of the adult mice with
hypomyelination also generated Mbp-expressing myelin, suggesting the
reprogrammed cells become functional oligodendrocytes in vivo.

The
three transcription factors reprogrammed about 20% of the cultured mouse
fibroblasts into cells with OPC properties. The OPCs could be expanded in
culture for at least five passages, and the set of three transcription factors
also was sufficient to reprogram mouse lung fibroblasts into OPCs.

In
the other paper, Marius Wernig and colleagues at Stanford used a group of
transcription factors that partially overlapped with those used by the Case
Western group to reprogram both mouse and rat fibroblasts into OPCs.

Wernig
is assistant professor of pathology at Stanford's Institute for Stem Cell
Biology and Regenerative Medicine.

The
Stanford team first used a pool of lentiviruses carrying 10
oligodendrocyte-specific transgenes to reprogram mouse embryonic fibroblasts
into OPCs. The team then shrank the cocktail to three transgenes-Sox10, Olig2
and zinc finger protein 536 (Zfp536)-sufficient to induce
effective reprogramming.

The
set of three transcription factors reprogrammed rat fibroblasts with about 15%
purity. The OPCs differentiated into both oligodendrocytes and astrocytes in
culture.

In
dorsal root ganglion neurons with extended axon beds, coculture with the
reprogrammed OPCs gave rise to Mbp-expressing cells, suggesting the OPCs
effectively myelinated the axons.

Wernig
and colleagues used the same mouse model as the Case Western group but directly
injected the reprogrammed rat cells into the corpus callosum and cerebellum
brain sections of neonatal mice. Administration of doxycycline in drinking
water induced sustained transgene expression and led to the formation of
Mbp-expressing myelin around neurofilaments.

"Our
next steps are to utilize this technology on human cells to produce
patient-specific, functional oligodendrocyte progenitor cells and
oligodendrocytes for use in understanding and treating human disorders of
myelin," Tesar said.

Wernig's
team also plans to look into ways to convert human fibroblasts into induced
OPCs.

Mike
Gresser, CSO of the Myelin Repair Foundation, said, "These
human OPCs will be critical for in vitro studies of myelination and to
evaluate myelin repair drug candidates in the human brain."

The
Myelin Repair Foundation funded the work at
Case Western.

Frank
Edenhofer, head of stem cell engineering at the University of Bonn's Institute of
Reconstructive Neurobiology, told SciBX, "The most critical point
for the clinical realization of the approach is the adaptation to the human
system. I expect this to be doable; however, it might need a different
combination of transcription factors or other stimuli such as microRNA."

Cell source
alternatives

An open question is whether the direct
lineage conversion method will be better than available donor or stem cell
sources.

"The
key question for this approach is: What is the advantage of creating oligodendrocytes
in this way specifically?" said Sheng Ding, professor of pharmaceutical
chemistry at the University of California, San Francisco and a senior
investigator at the Gladstone Institutes. "This is key
because there are already other methods to create oligodendrocytes. We need to
determine whether this would be the most practical, safe and useful method to
make large masses of oligodendrocyte progenitors in vitro for clinical
use."

Malin
Parmar, associate professor of developmental neurobiology at Lund University, thinks direct
conversion could indeed be the optimal approach. "The advantage of direct
conversion is that you bypass the pluripotent stage and thus avoid the risk of
tumor formation or overgrowth due to uncontrolled proliferation after transplantation.
Another advantage is that it is generally quicker and easier, which also means
cheaper" than other approaches, she said. "Quicker is good because
the less time cells are kept in vitro, the fewer things can go wrong and
the fewer steps needed for quality control."

Ding
said bypassing the stem cell step does reduce the level of risk of
tumorigenesis but added that "there is always a risk when transforming
cells. The transformation can cause them to become tumorigenic or unstable, and
you can introduce different dangers through gene modification. This still uses
a gene integration method and carries the associated dangers" such as
cancer or unexpected toxicities.

Both
Ding and Edenhofer were concerned about the efficiency of the direct reprogramming
methods.

"Based
on the data reported, the conversion appears not to result in a homogeneous
induced oligodendrocyte progenitor population. Instead, reprogrammed induced
oligodendrocyte progenitor populations might represent a quite heterogeneous
population of fully and partially reprogrammed cells," said Edenhofer.

Ding
added, "The fact that the genetic reprogramming only reprograms a small
percentage of cells to the target cell type is a problem because it may not
generate a functional and useful population, and the contamination with cells
that are not fully reprogrammed could be dangerous."

Wernig
told SciBX that his team is working to improve the reprogramming process
to yield higher numbers of OPCs.

Therapeutic
remyelination

Regardless of where the oligodendrocytes
come from, the therapeutic effect in specific human diseases remains to be
shown.

One
concern, said Gresser, is that "inadequate quantities of oligodendrocyte
progenitor cells in the brains of MS patients might not be what limits myelin repair
of demyelinated lesions. It is possible that inadequate remyelination is due to
factors present in or near the lesions that limit the ability of the
oligodendrocyte progenitor cells that are there to proliferate and/or
differentiate into myelination oligodendrocytes that properly myelinate
demyelinated axons."

He
added, "It should not be taken for granted that introducing human
oligodendrocyte progenitor cells into the brain of an MS patient will by itself
result in good myelin repair."

Tesar
told SciBX that the Myelin Repair Foundation filed for a patent covering
the direct cell fate conversion of somatic cells into OPCs and
oligodendrocytes, which was assigned to Case Western. The IP is available for
licensing.

Wernig
said that two years ago, Stanford filed a patent application for the direct
conversion of fibroblasts to neurons with a possibility to also induce
oligodendrocytes using the same methods. He said that for undisclosed reasons,
a patent was never issued.

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